The long-term objective of this project is to understand the structure, function and mechanism of the oligosaccharyltransferase (OST). The OST transfers high mannose oligosaccharides onto the asparagines residues of nascent proteins in the lumen of the rough endoplasmic reticulum. During the proposed funding period, particular emphasis will be placed on a biochemical, structural and molecular characterization of the mammalian and fungal OST complexes. The canine OST has been resolved into multiple forms that differ with respect to subunit composition and enzyme activity. Interestingly, mammalian orthologues of two of the yeast OST subunits (Stt3p and Ost3p) are encoded by multiple genes (STT3-A and STT3-B; N33 and lAP). Multiple tissue Northern blots reveal tissue-specific differences in the relative expression of these mRNAs. One objective of this project is to elucidate the role of OST isoform diversity and to understand how differences in subunit composition effect enzyme activity. Kinetic experiments suggest that the oligosaccharyltransferase has distinct catalytic and regulatory binding sites for the dolichol-linked oligosaccharide. Novel assays and biochemical probes will be developed to determine the locations of the binding sites for the dolichol-linked oligosaccharide donors and the N-X-S/T acceptors in the nascent polypeptide. Yeast genetics methods will be used to identify important amino acid residues in the tripeptide-binding site. A third objective of this project will be to obtain a low to medium resolution structure of the yeast and mammalian OST complexes by 3-dimensional reconstruction of images that have been obtained by cryoelectron microscopy. Antibodies and chemical probes will be used to map the locations of substrate binding sites and cytosolic and lumenal domains onto the OST structure. The fourth objective of this project is to determine the molecular basis for novel subtypes of Congenital Disorders of Glycosylation. Fibroblast cell lines will be screened for defects in dolichol-linked oligosaccharide metabolism and deficiencies in oligosaccharyltransferase activity. [unreadable] [unreadable]